Participation of mouse DNA polymerases iota, eta, and rev1 in translesion synthesis of carcinogen induced DNA adducts and carcinogenesis.

Recent advances in understanding the molecular mechanisms of mutagenesis indicate that most mutations are dependent on the activity of translesion synthesis DNA polymerases. The impact of reducing the level of these polymerases on mutagenesis and carcinogenesis in mouse models is poorly defined. Using knock out strategies we were able to remove polymerase eta (pol ç), and polymerase iota (pol é) and pol eta/pol iota from the mouse and lower REV1 in the mouse lung. This dissertation reports the changes in UV-induced carcinogenesis and mutagenesis that were observed. Mutagenesis data in pol iota deficient cells clearly indicate pol iota as a mutagenic TLS polymerase in UV lesion bypass. Pol iota removal effectively lowered the mutational frequency in both pol eta null and wild-type backgrounds. Unexpectedly, after Hprt mutant screening, pol eta and pol iota deficient cells were found to participate in UV lesion bypass in a strand-specific manner. This suggests that not only does bypass of photoproducts occur but occurs in an asymmetrical fashion, with preference of polymerases for leading or lagging strand. To examine the hypothesis that reducing the mutagenic load will reduce the incidence of cancer, we examined how pol iota status contributes to carcinogenesis. Despite the fact that pol iota was mutagenic in bypass of UV induced lesions, its removal accelerated cancer formation in the pol eta null background. The mechanisms behind this tumor suppressor function remain elusive, but indicate pol iota may have additional cellular roles in conjunction with its polymerase activity. Examining the hypothesis that reducing the mutagenic load will reduce the incidence of cancer, we developed strategies to reduce REV1 in mouse models of lung carcinogenesis. Endogenous REV1 mRNA in the lung was effectively lowered with the use of gene delivery of REV1 targeting ribozyme. This reduction was found to effectively decrease the multiplicity of B[a]P-induced lung tumors. This reduction did not affect the size or types of tumors induced suggesting inhibition of cancer formation occurred at the initiation step. Collectively, these data yield insight into the molecular mechanisms of mutagenesis that induce cancer formation

Unconventional Ubiquitin Recognition by the Ubiquitin-Binding Motif within the Y-Family DNA Polymerases ι and Rev1

Translesion synthesis is an essential cell survival strategy to promote replication after DNA damage. The accumulation of Y family polymerases (pol) ι and Rev1 at the stalled replication machinery is mediated by the ubiquitin-binding motifs (UBMs) of the polymerases and enhanced by PCNA monoubiquitination. We report the solution structures of the C-terminal UBM of human pol ι and its complex with ubiquitin. Distinct from other ubiquitin-binding domains, the UBM binds to the hydrophobic surface of ubiquitin centered at L8. Accordingly, mutation of L8A, but not I44A, of ubiquitin abolishes UBM binding. Human pol ι contains two functional UBMs, both contributing to replication foci formation. In contrast, only the second UBM of Saccharomyces cerevisiae Rev1 binds to ubiquitin and is essential for Rev1-dependent cell survival and mutagenesis. Point mutations disrupting the UBM-ubiquitin interaction also impair the accumulation of pol ι in replication foci and Rev1-mediated DNA damage tolerance in vivo.National Institute of General Medical Sciences (U.S.) (Grant GM-079376)American Cancer Society. Research ProfessorshipNational Institute of Environmental Health Sciences (Grant P30 ES-002109

Inaccurate DNA Synthesis in Cell Extracts of Yeast Producing Active Human DNA Polymerase Iota

Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn2+ ions, can bypass some DNA lesions and misincorporates “G” opposite template “T” more frequently than incorporates the correct “A.” We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of “G” versus “A” method of Gening, abbreviated as “misGvA”). We provide unambiguous proof of the “misGvA” approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The “misGvA” activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts

Implication of 4E-BP1 protein dephosphorylation and accumulation in pancreatic cancer cell death induced by combined gemcitabine and TRAIL

Pancreatic cancer cells show varying sensitivity to the anticancer effects of gemcitabine. However, as a chemotherapeutic agent, gemcitabine can cause intolerably high levels of toxicity and patients often develop resistance to the beneficial effects of this drug. Combination studies show that use of gemcitabine with the pro-apoptotic cytokine TRAIL can enhance the inhibition of survival and induction of apoptosis of pancreatic cancer cells. Additionally, following combination treatment there is a dramatic increase in the level of the hypophosphorylated form of the tumour suppressor protein 4E-BP1. This is associated with inhibition of mTOR activity, resulting from caspase-mediated cleavage of the Raptor and Rictor components of mTOR. Use of the pan-caspase inhibitor Z-VAD-FMK indicates that the increase in level of 4E-BP1 is also caspase-mediated. ShRNA-silencing of 4E-BP1 expression renders cells more resistant to cell death induced by the combination treatment. Since the levels of 4E-BP1 are relatively low in untreated pancreatic cancer cells these results suggest that combined therapy with gemcitabine and TRAIL could improve the responsiveness of tumours to treatment by elevating the expression of 4E-BP1

Influence of local sequence context on damaged base conformation in human DNA polymerase ι: molecular dynamics studies of nucleotide incorporation opposite a benzo[a]pyrene-derived adenine lesion

Human DNA polymerase ι is a lesion bypass polymerase of the Y family, capable of incorporating nucleotides opposite a variety of lesions in both near error-free and error-prone bypass. With undamaged templating purines polymerase ι normally favors Hoogsteen base pairing. Polymerase ι can incorporate nucleotides opposite a benzo[a]pyrene-derived adenine lesion (dA*); while mainly error-free, the identity of misincorporated bases is influenced by local sequence context. We performed molecular modeling and molecular dynamics simulations to elucidate the structural basis for lesion bypass. Our results suggest that hydrogen bonds between the benzo[a]pyrenyl moiety and nearby bases limit the movement of the templating base to maintain the anti glycosidic bond conformation in the binary complex in a 5′-CAGA*TT-3′ sequence. This facilitates correct incorporation of dT via a Watson−Crick pair. In a 5′-TTTA*GA-3′ sequence the lesion does not form these hydrogen bonds, permitting dA* to rotate around the glycosidic bond to syn and incorporate dT via a Hoogsteen pair. With syn dA*, there is also an opportunity for increased misincorporation of dGTP. These results expand our understanding of the versatility and flexibility of polymerase ι and its lesion bypass functions in humans

How evaluative pairings improve body dissatisfaction in adult women

$\bf Background$ Many young women are dissatisfied with their bodies. This study investigated the effect on current body dissatisfaction levels of a newly developed evaluative conditioning procedure that paired self-similar and self-dissimilar images of bodies with positive and neutral affective images, respectively. We hypothesized that learning the contingency that self-similar bodies predict positive affectivity is one process that could aid in explaining how these procedures function. $\bf Methods$ Adult women without disordered eating pathology participated in an online experiment with random assignment to an intervention or a control condition. All participants initially rated body images in self-similarity and were subsequently asked to categorize positive and neutral images by valence as quickly and accurately as possible. In the intervention condition, self-similar bodies systematically preceded positive images, and self-dissimilar images preceded neutral images, creating a $\textit {similar body}$ $\rightarrow$ $\it positive$ contingency. Pairings in the control condition were unsystematic such that no contingency was present. We measured categorization latencies and accuracies to infer contingency learning as well as current body dissatisfaction immediately before and after exposure to the pairings. All participants further completed measures of trait body image concerns and disordered eating psychopathology at baseline, which we examined as moderators of an expected relation between condition assignment, contingency learning, and body dissatisfaction improvements. $\bf Results$ We analyzed data from N = 173 women fulfilling the inclusion criteria. Moderated mediation analyses showed that assignment to the intervention (vs. control) condition predicted increased $\textit {similar body}$ $\rightarrow$ $\it positive$ contingency learning, which in turn predicted improved body dissatisfaction post-intervention, but only among women with higher pre-existing trait body image concerns or disordered eating levels. $\bf Conclusions$ The findings point toward the relevancy of further exploring the utility of pairing procedures. $\textit {Similar body}$ $\rightarrow$ $\it positive$ contingency learning predicted improved body dissatisfaction in individuals with normatively high body image concerns, which suggests pairing procedures could help inform future research on reducing body dissatisfaction